重组CHO细胞C_(28)株S基因序列的测定及分析

目的测定重组CHO细胞C28株S基因序列,研究其遗传稳定性,并与已全基因序列测定的乙型肝炎病毒的S基因序列进行比较分析,预测和揭示现有疫苗株对当前疾病流行株的防病效果。方法从C28株中选取第22代、24代2、5代2、7代、28代2、9代3、0代、31代3、2代3、3代和34代细胞,根据GenBank中C基因型adr亚型乙型肝炎病毒的全基因序列设计引物。采用酚-氯仿法抽提CHO细胞基因组DNA,用PCR法扩增各代次细胞的S基因,回收700 bp左右的目的片段,克隆至pMD18-T载体上进行序列测定。利用生物学软件MEGA4.1和BioEdit进行S基因序列同源性分析,绘制系统进化树,分析与其他HBV病毒株S基因的同源性。应用实验动物测定C28株生产的重组乙型肝炎疫苗的效价。结果 C28株十一个代次之间S基因序列核苷酸和氨基酸同源性均为100%;C28株十一个代次S基因与其他病毒株S基因比较,与C基因型乙型肝炎病毒同源性最高,与其他基因型乙型肝炎病毒的核苷酸同源性达91.4%～95.1%,氨基酸同源性达84.5%～93.3%。免疫NIH小鼠结果显示5批重组乙型肝炎疫苗的效价均符合标准。结论 C28株S基因在传代及保存过程中具有较高的稳定性,对当前疾病流行株有较好的防病效果。

Sequencing and characteristics of S gene in recombinant CHO cell C28 Strain

Objective To determine the sequences of S gene in recombinant CHO cell C28 strain,study the genetic stability,and compare with the information which have include the complete genome sequences for hepatitis B virus S gene,forecast and warn the disease prevention effect on the current epidemic disease strains.Methods Selecting the cells of 22nd 24th,25th,27th,28th,29th,30th,31st,32nd,33rd and 34th generation from the C28 strain.Designing primers according to the complete genome sequences for hepatitis B virus in C genotype and adr subtype in GenBank and the principle of complementary base pairing.Extracting genomic DNA by hydroxybenzene-chloroform method from CHO cell,amplifying the specific fragment by PCR,recovering the gene fragments at lengths of 700 bp and cloning into pMD18-T vector.The genomic DNAs were sequenced,and the results were compared with those of S gene of other HBV strains,based on which a phylogenetic tree was generated to analyze the homologies of S gene to other strains by bio-software MEGA4.1 and BioEdit,and the potency was determined by laboratory animal.Results Genetic analysis revealed that the nucleotide and amino acid homologies of S gene among the C28 strain of eleven generations were 100%.In comparison with the other HBV stains,it was found that the nucleotide and amino acid homologies of S gene with C genotype was highest;the nucleotide homologies with other genotype HBV strains were 91.4%-95.1%,while the amino acid homologies were 84.5%-93.3%.The potency induced in mice by the five batches of recombinant hepatitis B vaccine were high.Conclusions CHO cells C28 strain S gene during subculture has a high stability,and the current epidemic strains have better control efficacy.